Construction of the Five-point Mutant in Novel Aspartokinase and Its Enzymatic Characterization from <i>Corynebacterium pekinense</i>

WANG Zheren; LIU Xiaoting; FAN Zhanqing; WANG Yanan; WEI Zhen; GAO Xin; FANG Li; MIN Weihong

doi:10.13386/j.issn1002-0306.2021010071

Volume 42 Issue 16

Aug. 2021

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Science and Technology of Food Industry > 2021 > 42(16): 112-118. > DOI: 10.13386/j.issn1002-0306.2021010071

WANG Zheren, LIU Xiaoting, FAN Zhanqing, et al. Construction of the Five-point Mutant in Novel Aspartokinase and Its Enzymatic Characterization from Corynebacterium pekinense[J]. Science and Technology of Food Industry, 2021, 42(16): 112−118. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021010071.

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Construction of the Five-point Mutant in Novel Aspartokinase and Its Enzymatic Characterization from Corynebacterium pekinense

National Engineering Laboratory for Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China

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Received Date: January 12, 2020
Available Online: June 20, 2021

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Abstract

Abstract

Purpose: To improve the activity of the first key allosteric enzyme-aspartate kinase (AK) in the aspartate amino acid synthesis pathway, and to weaken or remove the terminal products lysine (Lys) and threonine (Thr) synergistic feed back inhibition of AK. Methods: On this basis of the four-point mutant T379N/A380C/G171I/Y198N AK(NCIN AK) of Corynebacterium pekinense, it was found that the highly conserved mutation sites of Gly295 was bound with inhibitor Thr by hydrogen bonds. The site of Gly295 were selected for site-directed mutation. The plasmid was extracted and transformed into E. coli competent BL21 to induce expression. High-throughput screening was used to obtain mutant strains with significantly improved enzyme activity. Enzyme kinetic study and characterization of enzyme properties were performed on the wild type(WT) and the five-point mutant T379N/A380C/G171I/Y198N/G295L AK(NCINL AK). Results: The five-point mutant NCINL AK was successfully constructed, and its maximum reaction rate V_max was 259 U/(mg·min). The K_m value of the Michaelis constant of the mutant reduced from 3.44 mmol/L to 0.93 mmol/L, and the substrate affinity was enhanced compared with the wild type enzyme. The hill coefficient n value was reduced from 2.73 to 1.21, and the positive synergy was decreased. The optimum temperature of NCINL AK increased from 25 ℃ to 28 ℃, the optimum pH value increased from 8.0 to 8.5, and the half-life was extended from 4.7 h to 5.2 h. Compared with the wild type WT AK, the five-point mutant NCINL AK had different inhibitory effects at different substrate inhibitors with concentration of 0.2, 1.0, 5.0, 10.0 mmol/L and even the inhibitory effect of Lys was completely lifted, and it had an activation effect under the condition of 10 mmol/L Thr. Conclusion: In this experiment, the five-point mutant NCINL AK with enzyme activity increased by 87.20 times was obtained, and the enzymatic properties were also significantly improved. Within a certain inhibitor concentration range, the feedback-inhibition effect of Lys on AK was basically lifted, and to a certain extent releasing Thr's feedback inhibition provided the possibility for the accumulation of large amounts of aspartate amino acids and provided reference for constructing high-yield aspartate amino acid strains. It was possible for us to get the pollution-free and high-efficiency amino acids such as methionine (Met).
- Corynebacterium pekinense,
- aspartate kinase,
- five-point mutant,
- enzyme kinetics,
- enzymatic properties

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References

[1]	郭永玲. 北京棒杆菌(Corynebacterium pekinense)天冬氨酸激酶的定点突变及突变株酶学性质表征[D]. 长春: 吉林农业大学, 2014.
[2]	Manjasetty B A, Chance M R, Burley S K, et al. Crystal structure of Clostridium acetobutylicum aspartate kinase (CaAk): An important allosteric enzyme for amino acids production[J]. Biotechnology Reports,2014,3:73−85. doi: 10.1016/j.btre.2014.06.009
[3]	任军, 闵伟红, 詹冬玲, 等. 天冬氨酸激酶突变体G277K中AK基因的克隆表达及酶学性质表征[J]. 食品科学,2014,35(11):149−154. doi: 10.7506/spkx1002-6630-201411030
[4]	Mas D C, Curien G, Robert G M, et al. A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase[J]. The Plant Cell Online,2006,18(7):1681~1692.
[5]	Chen Z, Meyer W, Rappert S, et al. Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacteriumglutamicum for lysine production[J]. Applied and Environmental Microbiology,2011,77(13):4352−4360. doi: 10.1128/AEM.02912-10
[6]	Kotaka M, Ren J, Lockyer M, et al. Structures of R-and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine[J]. The Journal of Biological Chemistry,2006,281(42):31544−31552.
[7]	Kikuchi Y, Kojima H, Tanaka T. Mutational analysis of the feedback sites of lysine-sensitive aspartokinase of Escherichia coli[J]. FEMS Microbiology Letters,2006,173(1):211−215.
[8]	Dong X, Quinn P J, Wang X. Metabolic engineering of Escherichia coli and Corynebacteriumglutamicum for the production of L-threonine.[J]. Biotechnology Advances,2011,29(1):11−23. doi: 10.1016/j.biotechadv.2010.07.009
[9]	Renaud D, David C, Robin A Y, et al. The many faces of aspartate kinases[J]. Archives of Biochemistry and Biophysics,2012,519(2):186−193. doi: 10.1016/j.abb.2011.10.016
[10]	Yoshida A, Tomita T, Kurihara T, et al. Structural insight into concerted inhibition of α2β2-Type aspartate kinase from Corynebacterium glutamicum[J]. Journal of Molecular Biology,2007,368(2):521−536. doi: 10.1016/j.jmb.2007.02.017
[11]	Curien G, Laurencin M, Mylène Robert-Genthon, et al. Allosteric monofunctional aspartate kinases from Arabidopsis[J]. 2007, 274(1): 164-176.
[12]	Paris S, Viemon C, Curien G, et al. Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine[J]. Journal of Biological Chemistry,2003,278(7):5361−5366. doi: 10.1074/jbc.M207379200
[13]	Han C J, Fang L, Liu C L, et al. Construction of novel aspartokinase mutant A380I and its characterization by molecular dynamics simulation[J]. Molecules,2018,23(12):3379. doi: 10.3390/molecules23123379
[14]	Tsujimoto M, Yoshida A, Shimizu T, et al. Aspartate kinase involved in 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis[J]. Journal of the Agricultural Chemical Society of Japan,2016,80(11):1−9.
[15]	赵智, 刘阳剑, 王宇, 等. 抗反馈抑制的天冬氨酸激酶基因在钝齿棒杆菌中的表达[J]. 微生物学报,2005,45(4):530−533. doi: 10.3321/j.issn:0001-6209.2005.04.009
[16]	秦天宇. 代谢工程改造谷氨酸棒杆菌生产L-甲硫氨酸[D]. 无锡: 江南大学, 2014.
[17]	Shaul O, Galili G. Threonine overproduction in transgenic tobacco plants expressing a mutant desensitized aspartatekinase of Escherichia coli[J]. Plant Physiology,1992,100(3):1157−1163. doi: 10.1104/pp.100.3.1157
[18]	Ohnishi J, Mitsuhashi S, Hayashi M, et al. A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant[J]. Applied Microbiology and Biotechnology,2002,58(2):217−223. doi: 10.1007/s00253-001-0883-6
[19]	Chen Z, Rappert S, Sun J, et al. Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production[J]. Journal of Biotechnology,2011,154(4):248−254. doi: 10.1016/j.jbiotec.2011.05.005
[20]	Curien G, Laurencin M, Mylène R G, et al. Allosteric monofunctional aspartate kinases from arabidopsis[J]. FEBS Journal,2007,274(1):164−176. doi: 10.1111/j.1742-4658.2006.05573.x
[21]	Yoshida A, Tomita T, Kuzuyama T, et al. Mechanism of concerted inhibition of α2β2-type hetero-oligomeric aspartate kinase from Corynebacterium glutamicum[J]. Journal of Biological Chemistry,2010,285(35):27477−27486. doi: 10.1074/jbc.M110.111153
[22]	Han C J, Liu S M, Liu C L, et al. The mutant T379L of novel aspartokinase from Corynebacteriumpekinense: A combined experimental and molecular dynamics simulation study[J]. Process Biochemistry,2019,83:77−85. doi: 10.1016/j.procbio.2019.04.022
[23]	陈志杰, 王鹏, 詹冬玲, 等. 北京棒杆菌天冬氨酸激酶突变体A380H的酶学性质[J]. 吉林大学学报(理学版),2017,55(5):1−8.
[24]	张芷睿, 陈晨, 韩彩静, 等. 北京棒杆菌天冬氨酸激酶突变体Q316P的酶学性质[J]. 微生物学报,2018,58(5):842−850.
[25]	Min W H, Li H Y, Li H M, et al. Characterization of aspartate kinase from Corynebacterium pekinense and the critical site of Arg169[J]. International Journal of Molecular Sciences,2015,16(12):28270−28284. doi: 10.3390/ijms161226098
[26]	Li C C, Yang M J, Liu L, et al. Mechanistic insight into the allosteric regulation of Pseudomonas aeruginosa aspartate kinase[J]. Biochemical Journal,2018,475:1107−1119. doi: 10.1042/BCJ20170829
[27]	Gao Y N, Fang L, Min W H, et al. Enzymatic characterization and molecular mechanism of novel aspartokinase mutant M372I/T379W from Corynebacterium pekinense[J]. RSC Advanes,2019:21344−21354.
[28]	Kalachova T, Janda M, Šašek V, et al. Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant[J]. Annals of Botany,2020,125(5):5.
[29]	魏丽茵, 吕天晓, 范甜, 等. 利用CRISPR/Cas9技术构建拟南芥IQM家族基因四突变体[J]. 科技视界,2020(12):166−168.
[30]	魏贞, 韩彩静, 高云娜, 等. 北京棒杆菌新型天冬氨酸激酶双突变株Y198N/D201M的构建及酶学性质表征[J]. 食品科学,2020,41(18):127−133. doi: 10.7506/spkx1002-6630-20190724-321

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